Exploring capabilities of elemental mass spectrometry for determination of metal and biomolecules in extracellular vesicles.

Extracellular vesicles (EVs) are increasingly recognized as crucial components influencing various pathophysiological processes, such as cellular homeostasis, cancer progression, and neurological disease. However, the lack of standardized methods for EV isolation and classification, coupled with ambiguity in biochemical markers associated with EV subtypes, remains a major challenge. This Trends article highlights the most common approaches for EV isolation and characterization, along with recent applications of elemental mass spectrometry (MS) to analyse metals and biomolecules in EVs obtained from biofluids or in vitro cellular models. Considering the promising capabilities of elemental MS, the article also looks ahead to the potential analysis of EVs at the single-vesicle and single-cell levels using ICP-MS. These approaches may offer valuable insights into individual characteristics of EVs and their functions, contributing to a deeper understanding of their role in various biological processes.

Elemental mass spectrometry to study metallo-transcriptomic changes during the in vitro degeneration of the retinal pigment epithelium.

Trace elements play crucial roles in cellular biology. Their improper homeostasis may contribute to the progress of eye diseases, exacerbated during ageing. The retinal pigment epithelium (RPE) is progressively deteriorated during age-related neurodegeneration and metal homeostasis may be compromised. In this study, elemental mass spectrometry (MS) was combined with cellular and molecular biology techniques to identify changes in trace elements during the in vitro degeneration of human RPE cells. Cells were collected at 21, 91, and 133 days and processed for RNA sequencing; Ca, Na, P, Mg, and Cu quantification by flow injection analysis and inductively coupled plasma-MS; and protein analysis by immunocytochemistry. Four-month-old RPE cultures showed depigmentation, impaired barrier function, and antioxidant protection, manifesting signs of epithelial-to-mesenchymal transition. Na and P significantly increased in the cytosol of degenerated RPE cells (from 15 ± 20 to 13495 ± 638 ng·µg-1 and from 30.6 ± 9.5 to 116.8 ± 16.8 ng·µg-1, respectively). Mg decreased in both the cytosol and insoluble fraction of cells (from 2.83 ± 0.40 to 1.58 ± 0.56 ng·µg-1 and from 247.57 ± 11.06 to 30 ± 8 ng·g-1, respectively), while P and Cu decreased in the insoluble fraction after 133 days in culture (from 9471 ± 1249 to 4555 ± 985 ng·µg-1 and from 2251 ± 79 to 1054 ± 235 ng·g-1, respectively), along with changes in metal-dependent antioxidant enzymes and Cu transporters. This RPE model reflected metal homeostatic changes, providing additional perspectives on effects of metal regulation during ageing.

Single Cell-ICP-ToF-MS for the Multiplexed Determination of Proteins: Evaluation of the Cellular Stress Response.

An automated and straightforward detection and data treatment strategy for the determination of the protein relative concentration in individual human cells by single cell-inductively coupled plasma-time-of-flight mass spectrometry (sc-ICP-ToF-MS) is proposed. Metal nanocluster (NC)-labeled specific antibodies for the target proteins were employed, and ruthenium red (RR) staining, which binds to the cells surface, was used to determine the number of cell events as well as to evaluate the relative volume of the cells. As a proof of concept, the expression of hepcidin, metallothionein-2, and ferroportin employing specific antibodies labeled with IrNCs, PtNCs, and AuNCs, respectively, was investigated by sc-ICP-ToF-MS in human ARPE-19 cells. Taking into account that ARPE-19 cells are spherical in suspension and RR binds to the surface of the cells, the Ru intensity was related to the cell volume (i.e., the cell volume is directly proportional to (Ru intensity)3/2), making it possible to determine not only the mass of the target proteins in each individual cell but also the relative concentration. The proposed approach is of particular interest in comparing cell cultures subjected to different supplementations. ARPE-19 cell cultures under two stress conditions were compared: a hyperglycemic model and an oxidative stress model. The comparison of the control with treated cells shows not only the mass of analyzed species but also the relative changes in the cell volume and concentration of target proteins, clearly allowing the identification of subpopulations under the respective treatment.

Homeostatic alterations related to total antioxidant capacity, elemental concentrations and isotopic compositions in aqueous humor of glaucoma patients.

Glaucoma is a multifactorial eye disease, characterized by progressive optic neurodegeneration. Elevation of the intraocular pressure is the main risk factor for glaucoma and is a consequence of an imbalance in the aqueous humor hydrodynamics, the physiology of which is influenced by the homeostatic equilibrium of essential elements, oxidative stress, and antioxidants. The aim of this work was to study local alterations in glaucomatous patients from two different, but connected, points of view: (i) the total antioxidant capacity (as an indicator of oxidative damage) and (ii) the concentration of mineral elements and their isotopic composition. Such objective was pursued using aqueous humor from patients diagnosed with pseudoexfoliation glaucoma (PEXG, n = 17) and primary open-angle glaucoma (POAG, n = 5) and age-matched control subjects (n = 16). The total antioxidant capacity (TAC) was examined in both aqueous humor and 60 serum samples (n = 20 controls, n = 20 for PEXG, and n = 20 for POAG), both showing higher TAC for the glaucoma population. The concentrations of the essential mineral elements (Cu, Fe, Mg, Na, P, and Zn) and the isotopic compositions of Cu and Zn were determined in aqueous humor using single-collector and multi-collector inductively coupled plasma-mass spectrometry, respectively. Significant differences were established for Mg and P levels when comparing the results for glaucomatous patients with those for the control population (p < 0.01 and p < 0.05 for Mg and P respectively, ANOVA and Kruskal-Wallis). The Zn isotopic composition was significantly shifted from that for the control population for PEXG patients. A significant difference in the isotopic composition of Zn was also established between the PEXG and POAG glaucoma cohorts.

Isotopically Enriched Tracers and Inductively Coupled Plasma Mass Spectrometry Methodologies to Study Zinc Supplementation in Single-Cells of Retinal Pigment Epithelium in Vitro.

Mass spectrometry-based techniques, such as inductively coupled plasma mass spectrometry (ICPMS) and laser ablation (LA) ICPMS, combined with an isotope pattern deconvolution mathematical tool are proposed for a better understanding of supplementation studies in cultured cells. An in vitro model of human retinal pigment epithelium (HRPEsv) cells was treated with different concentrations (0-150 µm Zn, 1 mL) of enriched stable isotope tracers of Zn in the form of sulfate and/or gluconate. Supplementations with t68ZnSO4 or t70Zn-gluconate alone and in combination (1:1 molar ratio) were investigated to evaluate the exogenous contribution and distribution of Zn in the treated cells. In order to obtain not only the Zn concentration for a cell population (mineralized cells) but also single cell information about the contribution of exogenous Zn and their distribution within micrometer cells structures, LA-ICPMS was employed to directly analyze cryopreserved cells. natZn, t68Zn, and t70Zn molar fraction images obtained from cells and cell aggregates allowed confirming the uptake of exogenous Zn by HRPEsv cells, being t68Zn and t70Zn molar fractions close to 1 in the cell nuclei. Under the selected experimental conditions tested (24 h treatments), no significant differences were obtained in the Zn distribution depending on its chemical form.

Quantitative mapping of specific proteins in biological tissues by laser ablation-ICP-MS using exogenous labels: aspects to be considered.

Laser ablation (LA) coupled with inductively coupled plasma mass spectrometry (ICP-MS) is a versatile tool for direct trace elemental and isotopic analysis of solids. The development of new strategies for quantitative elemental mapping of biological tissues is one of the growing research areas in LA-ICP-MS. On the other hand, the latest advances are related to obtaining not only the elemental distribution of heteroatoms but also molecular information. In this vein, mapping of specific proteins in biological tissues can be done with LA-ICP-MS by use of metal-labelled immunoprobes. However, although LA-ICP-MS is, in principle, a quantitative technique, critical requirements should be met for absolute quantification of protein distribution. In this review, progress based on the use of metal-labelled antibodies for LA-ICP-MS mapping of specific proteins is reported. Critical requirements to obtain absolute quantitative mapping of specific proteins by LA-ICP-MS are highlighted. Additionally, illustrative examples of the advances made so far with LA-ICP-MS are provided. Graphical abstract In the proposed critical review, last advances based on the use of metal-labelled antibodies and critical requirements for LA-ICP-MS quantitative mapping of specific proteins are tackled.

Bimodal determination of immunoglobulin E by fluorometry and ICP-MS by using platinum nanoclusters as a label in an immunoassay.

The authors describe the use of platinum nanoclusters (PtNCs) as bimodal labels in a competitive immunoassay for immunoglobulin E (IgE). Both fluorometry and inductively coupled plasma - mass spectrometry (ICP-MS) are used. Optimization of the PtNCs synthesis using lipoic acid as ligand was carried out. The time for synthesis and the effect of NaOH added to the PtNCs precursor mixture was optimized with the aim to obtain PtNCs with strong red fluorescence and low size dispersity. Maximal fluorescence was obtained at excitation/emission wavelengths of 455/620 nm. The average diameter (1.5 nm) and crystal structure (face-centered cubic structure) of the PtNCs were determined by HR-TEM. It was calculated that each PtNC contains 116 Pt atoms at average. Labelling of the antibody (Ab) against IgE with PtNCs was optimized in terms of recognition capabilities and fluorescence intensity. A molar ratio (Ab:PtNCs) of 1:11 is found to be best. A competitive immunoassay for IgE was developed and detection was carried out by using both ICP-MS (by measuring 195Pt) and fluorometry. The limit of detection (LOD) of the fluoroimmunoassay is 0.6 ng mL-1 of IgE. The LOD of the ICP-MS method is as low as 0.08 ng mL-1. The method was evaluated by analyzing four (spiked) serum samples by ICP-MS. No sample pretreatment excepting dilution is needed. Results compared favorably with those obtained by a commercial ELISA kit. Graphical abstract Schematic representation of the bimodal quantification (fluorescence and ICP-MS) of immunoglobulin E (Ig E) in human serum using antibody against human Ig E, labelled with several platinum nanoclusters (NCs) as immunoprobe. Elemental mass spectrometry (MS) allows high amplification of the signal because of the high number of platinum atoms per nanocluster (~116 Pt/NC).

Fluorescent silver nanoclusters as antibody label in a competitive immunoassay for the complement factor H.

Silver nanoclusters (AgNCs) were investigated as labels for the development of a fluoroimmunoassay for the complement factor H (CFH). The reductive one-pot synthesis of AgNCs using lipoic acid as a ligand was optimized by varying the concentration of NaBH4, the temperature and the reaction time. The average diameter and crystal structure of the AgNCs (which display red fluorescence) were determined by HR-TEM. The silver concentration was quantified by ICP-MS. Labelling of the antibody against CFH with AgNCs was optimized. The antibody was labeled with the AgNCs without compromising the recognition capabilities of the antibody. A competitive fluoroimmunoassay was then developed. Fluorescence is measured at excitation/emission maxima of 430/660 nm. The assay has a 0.4 ng mL-1 detection limit and a linear range that extends from 1.2 to 23 ng mL-1. The results compare favorably with those obtained by a commercial ELISA kit. The method was applied to the determination of CFH in spiked human serum. Graphical abstract Schematic presentation of the method for the determination of complement factor H (CFH) protein in human blood by using a CFH-antibody labelled with fluorescent silver nanoclusters.

Quantitative Imaging of Specific Proteins in the Human Retina by Laser Ablation ICPMS using Bioconjugated Metal Nanoclusters as Labels.

A sensitive methodology using antibody-conjugated gold nanoclusters (AuNCs) was developed for the quantitative bioimaging of specific proteins in biological tissues by laser ablation (LA) coupled to inductively coupled plasma-mass spectrometry (ICPMS). Determination of metallothioneins (MT1/2 protein isoforms) images in human retina tissue sections was carried out as a proof of concept. AuNCs used as label were conjugated to the selected antibody through carbodiimide coupling. A stoichiometry of AuNCs/available antibody of 1:1 was obtained. The high amplification provided by AuNC labels allowed for obtaining the distribution of MT1/2 in the neurosensory retina layers (5 µm thick sections) by LA-ICPMS. Elemental images of 197Au+ were quantified with gelatin matrix-matched standards and then converted to 2D quantitative images of MT1/2 concentration. For validation purposes, average concentrations of MT1/2 obtained in the human retinal layers by LA-ICPMS were successfully compared with those obtained with a commercial ELISA kit.

Iron bioavailability from supplemented formula milk: effect of lactoferrin addition.

PURPOSE: In this work, the absorption and/or bioavailability of iron from two chemical species, 57Fe-Lf (apo-lactoferrin) complex and 57FeSO4 at low and high dose, and in Lf excess were investigated in lactating wistar rats. METHODS: The methodology used is based on the use of stable isotopes in combination with the approach "isotope pattern deconvolution" and ICP-MS for detection. This approach provides quantitative information about exogenous (57Fe) and endogenous iron (natFe) distribution in fluids and tissues in the iron-supplemented rat groups. RESULTS: The observed results with supplemented rats were compared with those found in rats receiving maternal feeding. Interestingly, differences were found between groups in iron for transport and storage compartments, but not in the functional one, depending upon the dose of iron administered and the chemical species. CONCLUSION: Considering the results obtained, supplementation with iron salts in excess of Lf appears to be the best way of iron supplementation of formula milk.

Bioimaging of metallothioneins in ocular tissue sections by laser ablation-ICP-MS using bioconjugated gold nanoclusters as specific tags.

An immunohistochemical method is described to visualize the distribution of metallothioneins 1/2 (MT 1/2) and metallothionein 3 (MT 3) in human ocular tissue. It is making use of (a) antibodies conjugated to gold nanoclusters (AuNCs) acting as labels, and (b) laser ablation (LA) coupled to inductively coupled plasma - mass spectrometry (ICP-MS). Water-soluble fluorescent AuNCs (with an average size of 2.7 nm) were synthesized and then conjugated to antibody by carbodiimide coupling. The surface of the modified AuNCs was then blocked with hydroxylamine to avoid nonspecific interactions with biological tissue. Immunoassays for MT 1/2 and MT 3 in ocular tissue sections (5 µm thick) from two post mortem human donors were performed. Imaging studies were then performed by fluorescence using confocal microscopy, and LA-ICP-MS was performed in the retina to measure the signal for gold. Signal amplification by the >500 gold atoms in each nanocluster allowed the antigens (MT 1/2 and MT 3) to be imaged by LA-ICP-MS using a laser spot size as small as 4 µm. The image patterns found in retina are in good agreement with those obtained by conventional fluorescence immunohistochemistry which was used as an established reference method. Graphical abstract Gold nanoclusters (AuNCs) conjugated to a primary specific antibody serve as a label for amplified bioimaging of metallothioneins (MTs) by laser ablation coupled to inductively coupled plasma - mass spectrometry (ICP-MS) in human ocular tissue sections.

LOXL1 gene variants and their association with pseudoexfoliation glaucoma (XFG) in Spanish patients.

BACKGROUND: LOXL1 gene is the most important genetic risk factor known so far for pseudoexfoliation glaucoma (XFG). Our purpose was to evaluate the potential association of individual genetic variants of the lysyl oxidase-like 1 (LOXL1) gene and haplotypes with XFG in Spanish patients. METHODS: Blood samples were collected from a total of 105 Spanish patients with XFG and 200 healthy controls. The entire LOXL1 gene along with the promoter, coding and non-coding regions including the 5'- and 3'-untranslated regions, were sequenced using next-generation sequencing in 99 XFG patients. SNPs rs16958477 (promoter), rs1048661 (exon 1), rs3825942 (exon 1), rs2165241 (intron 1) and rs3522 (exon 7) in LOXL1 were genotyped by restriction fragment-length polymorphism (RFLP) in all Spanish control participants and in six additional XFG patients, and a case-control association study was performed. Comparisons of the allelic and genotypic frequencies were performed using standard χ(2) test with Bonferroni and Pearson corrections. Logistic regression analyses were permormed using Sigmaplot v11. Haplotypes frequencies were performed using HaploView 4.0. RESULTS: Sequencing of the LOXL1 gene in XFG participants identified a total of 212 SNPs, of which 49 exhibited allelic frequencies with significant differences between cases and controls, and 66 were not previously described. The allele frequencies of SNPs rs16958477, rs1048661, rs3825942, rs2165241, were significantly associated with an increased risk for XFG, however the SNP rs3522 was not. The haplotype frequencies of SNPs rs16958477, rs1048661, rs3825942 and rs2165241 and their association with XFG indicated that the CGGT haplotype, containing all four risk alleles, and the AGGT haplotype, which carries the protective allele of rs16958477 and three risk alleles of the other three SNPs, were significantly associated with XFG (p = 4.5×10(-6), and p = 8.8×10(-6)), conferring more than 2-fold increased disease susceptibility. CONCLUSIONS: SNPs of the LOXL1 gene are associated with XFG in the Spanish population. This information adds new support to the distinct risk association frequencies of LOXL1 alleles with XFG in Western European and Asian populations. Identification and validation of additional SNPs along the entire LOXL1 gene of XFG cases may provide insightful information on their potential role in the pathogenesis of this disease.

Quantitative selenium speciation by HPLC-ICP-MS(IDA) and simultaneous activity measurements in human vitreous humor.

A post-column isotope dilution analysis (IDA) methodology was applied to carry out quantitative speciation of selenium in human vitreous humor samples by size exclusion chromatography (SEC) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS). Two main selenium species detected by SEC-ICP-MS were found to be associated to protein complexes. The expected molecular weights for both selenium-bound complexes were confirmed by MALDI-TOF(MS) and the results matched well with the theoretical mass of a GPx monomer (M, 22 kDa) and tetramer (T, 88 kDa). The quantification of the two detected selenium-bound complexes by post-column IDA showed that the total content of selenospecies in vitreous humor was approximately 3.2 ± 1.8 ppb Se. Moreover, in most of the analyzed vitreous humor samples, the majority of the selenium was associated to higher molecular weight GPx biomolecules. In an attempt to assess if the enzymatic activity was associated with a given selenium-bound GPx protein, the antioxidant enzyme activity was assayed for the two separated GPx species. Only for GPx (T) was a linear relationship between activity and total Se concentration found by ICP-MS.

Quantitative bioimaging of trace elements in the human lens by LA-ICP-MS.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of Fe, Cu and Zn in cryostat sections of human eye lenses and for depth profiling analysis in bovine lenses. To ensure a tight temperature control throughout the experiments, a new Peltier-cooled laser ablation cell was employed. For quantification purposes, matrix-matched laboratory standards were prepared from a pool of human lenses from eye donors and spiked with standard solutions containing different concentrations of natural abundance Fe, Cu and Zn. A normalisation strategy was also carried out to correct matrix effects, lack of tissue homogeneity and/or instrumental drifts using a thin gold film deposited on the sample surface. Quantitative images of cryo-sections of human eye lenses analysed by LA-ICP-MS revealed a homogeneous distribution of Fe, Cu and Zn in the nuclear region and a slight increase in Fe concentration in the outer cell layer (i.e. lens epithelium) at the anterior pole. These results were assessed also by isotope dilution mass spectrometry, and Fe, Cu and Zn concentrations determined by ID-ICP-MS in digested samples of lenses and lens capsules.

Identification and characterization of a new pathologic mutation in a large Leber hereditary optic neuropathy pedigree.

BACKGROUND: Most patients suffering from Leber hereditary optic neuropathy carry one of the three classic pathologic mutations, but not all individuals with these genetic alterations develop the disease. There are different risk factors that modify the penetrance of these mutations. The remaining patients carry one of a set of very rare genetic variants and, it appears that, some of the risk factors that modify the penetrance of the classical pathologic mutations may also affect the phenotype of these other rare mutations. RESULTS: We describe a large family including 95 maternally related individuals, showing 30 patients with Leber hereditary optic neuropathy. The mutation responsible for the phenotype is a novel transition, m.3734A > G, in the mitochondrial gene encoding the ND1 subunit of respiratory complex I. Molecular-genetic, biochemical and cellular studies corroborate the pathogenicity of this genetic change. CONCLUSIONS: With the study of this family, we confirm that, also for this very rare mutation, sex and age are important factors modifying penetrance. Moreover, this pedigree offers an excellent opportunity to search for other genetic or environmental factors that additionally contribute to modify penetrance.

The stoichiometric transition from Zn6Cu1-metallothionein to Zn7-metallothionein underlies the up-regulation of metallothionein (MT) expression: quantitative analysis of MT-metal load in eye cells.

We examined the profiling of gene expression of metallothioneins (MTs) in human tissues from cadaver eyes with microarray-based analysis. All MT1 isoforms, with the exception of MT1B, were abundantly expressed in lens and corneal tissue. Along with MT1B, MT4 was not detected in any tissues. Antibodies to MT1/2 labeled the corneal epithelial and endothelial cells, whereas MT3 label the retinal ganglion cells. We studied the effects of zinc and cytokines on the gene expression of MT isoforms in a corneal epithelial cell line (HCEsv). Zinc exerted an up-regulation of the expression of MT isoforms, and this effect was further potentiated in the presence of IL1α or TNFα. Zinc also elicited a strong down-regulation of the expression of inflammatory cytokines, and this effect was blocked in the presence of TNFα or IL1α. The concentration of MTs, bound zinc, and the metal stoichiometry of MTs in cultured HCEsv were determined by mass spectrometry. The total concentration of MTs was 0.24 ± 0.03 µM and, after 24 h of zinc exposure, increased to 0.96 ± 0.01 µM. The combination of zinc and IL1α further enhanced the level of MTs to 1.13 ± 0.03 µM. The average metal stoichiometry of MTs was Zn(6)Cu(1)-MT, and after exposure to the different treatments, it changed to Zn(7)-MT. Actinomycin D blocked transcription, and cycloheximide attenuated synthesis of MTs in the presence or absence of zinc, suggesting transcriptional regulation. Overall the data provide molecular and analytical evidence on the interplay between zinc, MTs, and proinflammatory cytokines in HCEsv cells, with potential implications on cell-based inflammatory eye diseases.

Determination of endogenous trace elements in extracellular vesicles secreted by an in vitro model of human retinal pigment epithelium under oxidative stress conditions using ICP-MS.

The determination of endogenous Fe, Cu and Zn in exosomes (<200 nm extracellular vesicles) secreted by an in vitro model of the human retinal pigment epithelium (HRPEsv cell line) was carried out by inductively coupled plasma - mass spectrometry (ICP-MS). Results for cells treated with 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) inducing oxidative stress (OS) conditions were compared with non-treated (control) cells in order to evaluate possible differences in the metal composition between both groups. Three sample introduction systems were tested for ICP-MS analysis: a micronebulizer and two single cell nebulization systems (as total consumption set-ups), being found one of the single cell systems (operating in bulk mode) as the most suitable. Two protocols for the isolation of exosomes from cell culture media were investigated based on differential centrifugation and precipitation with a polymer-based reagent. Transmission electron microscopy measurements showed smaller and more homogeneous sizes (15-50 nm versus 20-180 nm size range) together with a higher particle concentration for exosomes purified by precipitation compared to differential centrifugation. However, it was observed that the contribution of polymer-based protocol to the Fe, Cu and Zn blank was significant as compared to the differential centrifugation protocol. Therefore, considering the low concentrations of the evaluated endogenous elements in exosomes from the HRPEsv cell line, the polymer-based precipitation method was discarded. When comparing metal levels in samples from control versus OS-treated HRPEsv cells, results for Fe and Cu were statistically similar. However, upregulation of Zn was found during OS conditions (11 versus 34 µg L-1 in control and OS-treatment, respectively), showing Zn depletion through secretory activity induced by OS, underlying the antioxidant ability of RPE cells.

Altered zinc homeostasis in a primary cell culture model of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) is progressively degenerated during age-related macular degeneration (AMD), one of the leading causes of irreversible blindness, which clinical hallmark is the buildup of sub-RPE extracellular material. Clinical observations indicate that Zn dyshomeostasis can initiate detrimental intracellular events in the RPE. In this study, we used a primary human fetal RPE cell culture model producing sub-RPE deposits accumulation that recapitulates features of early AMD to study Zn homeostasis and metalloproteins changes. RPE cell derived samples were collected at 10, 21 and 59 days in culture and processed for RNA sequencing, elemental mass spectrometry and the abundance and cellular localization of specific proteins. RPE cells developed processes normal to RPE, including intercellular unions formation and expression of RPE proteins. Punctate deposition of apolipoprotein E, marker of sub-RPE material accumulation, was observed from 3 weeks with profusion after 2 months in culture. Zn cytoplasmic concentrations significantly decreased 0.2 times at 59 days, from 0.264 ± 0.119 ng·µg-1 at 10 days to 0.062 ± 0.043 ng·µg-1 at 59 days (p < 0.05). Conversely, increased levels of Cu (1.5-fold in cytoplasm, 5.0-fold in cell nuclei and membranes), Na (3.5-fold in cytoplasm, 14.0-fold in cell nuclei and membranes) and K (6.8-fold in cytoplasm) were detected after 59-days long culture. The Zn-regulating proteins metallothioneins showed significant changes in gene expression over time, with a potent down-regulation at RNA and protein level of the most abundant isoform in primary RPE cells, from 0.141 ± 0.016 ng·mL-1 at 10 days to 0.056 ± 0.023 ng·mL-1 at 59 days (0.4-fold change, p < 0.05). Zn influx and efflux transporters were also deregulated, along with an increase in oxidative stress and alterations in the expression of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. The RPE cell model producing early accumulation of extracellular deposits provided evidences on an altered Zn homeostasis, exacerbated by changes in cytosolic Zn-binding proteins and Zn transporters, along with variations in other metals and metalloproteins, suggesting a potential role of altered Zn homeostasis during AMD development.

Relationship between Trace Elements and Matrix Metalloproteinases 2 and 9 and their Tissue Inhibitors in Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) constitutes around 5% of all thyroid cancers. Trace elements assessment has emerged as a useful strategy in the diagnostics of MTC combined with Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) analysis. The aim of this study was to compare the presence and content of trace elements (i.e., Copper (Cu), Zinc (Zn), Iron (Fe), and Manganese (Mn)) in MTC with respect to control samples and their potential relationship with markers of MTC in tissues. The study included 26 patients who had undergone thyroidectomy, due to the diagnosis of MTC and 17 patients as control. We combined tumour pathology and staging, immunohistochemical analysis of calcitonin, MMPs, and TIMPs, with analytical biochemistry using Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) to determine the levels of trace elements. No differences by MTC type for MMPs and their TIPMs, although strong TIMP-1 and TIMP-2 immunohistochemical expression of MTC were unveiled. Additionally, Zn, Fe, and Mn tended to be decreased, and Cu to be increased in samples presenting MTC with respect to controls. Moreover, Zn was the unique trace element which seemed to be correlated with MMPs and TIMPs. Trace elements such as Zn, Fe, and Mn are decreased in tissues affected by MTC. In addition, Zn may be the trace element which saves more relationship with the proportion and intensity of MMPs, being considered altogether useful biomarkers of MTC. We therefore suggest the analysis of novel and traditional markers of MTC as a novel approach in this pathology.

Determination and localization of specific proteins in individual ARPE-19 cells by single cell and laser ablation ICP-MS using iridium nanoclusters as label.

Single cell-inductively coupled plasma-mass spectrometry (sc-ICP-MS) and laser ablation (LA)-ICP-MS have been complementary employed to develop a comprehensive study of APOE and claudin-1 expression in ARPE-19 cells submitted to a glucose treatment (100 mM, 48 h) that induces oxidative stress conditions. Results were compared with control cells. The determination of the two proteins by ICP-MS was sequentially carried out using specific immunoprobes labelled with IrNCs that offer a huge amplification (1760 ± 90 atoms of Ir on average). A novel sample introduction system, the microFAST Single Cell set-up, was employed for sc-ICP-MS analysis. This introduction system resulted in a cellular transport efficiency of 85 ± 9% for ARPE-19 cells (91 ± 5% using a PtNPs standard). After the proper immunocytochemistry protocol with the specific IrNCs immunoprobes in cell suspensions (sc-ICP-MS), the mass of APOE and claudin-1 in individual ARPE-19 cells was obtained. Average detection limits per cell by sc-ICP-MS were 0.02 fg of APOE and 3 ag of claudin-1. The results of sample analyses obtained by sc-ICP-MS were validated with commercial ELISA kits. The distribution of both target proteins in individual cells (fixated in the chamber wall) was unveiled by LA-ICP-MS. The high amplification provided by the IrNCs immunoprobes allowed the identification of APOE and claudin-1 within individual ARPE-19 cells. High resolution images were obtained using a laser spot of 2 × 2 µm.